LABORATORY DIAGNOSIS OF PROTHROMBOTIC STATES

REGULATION OF COAGULATION Introduction Coagulation necessary for maintenance of vascular integrity Enough fibrinogen to clot all vessels What controls clotting process?

COAGULATION CASCADEVa/Xa/PL VIIIa/IXa/PL or VIIa/TF FXII FXIIa TF FVII FG F HMWK FXI FXIa VIIa/TF or FVIIa Ca +2 FIX FIXa Ca +2 VIIIVIIIa T VVa T Ca +2 FX FXa Ca +2 PT T Common Pathway Middle Components Surface Active Components INTRINSIC PATHWAYEXTRINSIC PATHWAY

COAGULATION INHIBITORS Tissue Factor Pathway Inhibitor (TFPI) Lipoprotein Associated Coagulation Inhibitor (LACI) Extrinsic Pathway Inhibitor (EPI) Complexes with Factors VIIa/TF/Xa; inactivates Xa Antithrombin III/Heparin Cofactor II/Heparin Binds and Inactivates Enzymes Protein C/Protein S/Thrombomodulin Cleaves & Inactivates Cofactors (Va & VIIIa) Plasminogen - 3º hemostasis Cleaves Fibrin

ANTICOAGULANT PROTEIN DEFICIENCY Disease entities Heterozygous Protein Deficiency Increased Venous Thrombosis Occasional Increased Arterial Thrombosis Warfarin Induced Skin Necrosis Homozygous Protein Deficiency Neonatal Purpura Fulminans Fibrinogenolysis Chronic DIC

ANTICOAGULANT PROTEIN DEFICIENCY Dominant Increased Venous Thrombosis Young Age of Thrombosis No Predisposing Factors to Thrombosis Increased Thrombin Generation Positive Family History Recessive No history of thrombosis No family history Neonatal Purpura Fulminans Increased Thrombin Generation

ACTIVATED PROTEIN C RESISTANCE 1st described by Dahlback, 1994 Hallmark: Failure of activated Protein C to prolong aPTT First noted in screening of plasma samples of patients with increased clotting Functional defect described before protein defect noted

ACTIVATED PROTEIN C RESISTANCE Bertina et al described genetic defect Mutation of Arg 506  Gln Named Factor V Leiden Found in > 98% of patients with APC Resistance

ACTIVATED PROTEIN C RESISTANCE Extremely common (5-20% of Caucasian population with mutation) Increases risk of venous thromboembolism (VTE) c. 4x in heterozygous form, more in homozygous Can exist in combination with other defects (protein C, protein S, ATIII, plasminogen In combination, has synergistic effect on other anticoagulant protein deficiencies

FACTOR V LEIDEN Normal procoagulant activity Inactivated slowly by activated protein C Leads to increased prothrombinase complex activity due to failure to remove factor Va Patients also display increased factor VIIIa/tenase activity

FACTOR Va INACTIVATION Factor VaiFVa APC Pro S PL PROTEIN C - MECHANISM OF ACTION

FACTOR VIIIa INACTIVATION Factor V is cofactor for Factor VIIIa inactivation Factor V Leiden unable to act as cofactor in VIIIa inactivation Therefore, increased VIIIa inactivation  increased tenase activity

FACTOR VIIIa INACTIVATION Factor VIIIaiFVIIIa APC Pro S PL Factor V PROTEIN C - MECHANISM OF ACTION

HYPERCOAGULABLE STATES Prothrombin G20210  A First described by Poort et al, 11/96 Mutation in 3’ non-coding sequence of prothrombin gene Northern European mutation (still being studied in non-European populations)

HYPERCOAGULABLE STATES Prothrombin G20210  A Mechanism of increased thrombosis unknown Increased prothrombin synthesis seen (> 115% of normal) Implicated in both arterial (stroke) and venous thrombosis as well as pregnancy-related thrombosis

HYPERCOAGULABLE STATES Hyperhomocysteinemia Inborn error of metabolism Leads to buildup of homocysteine via several pathways Homozygous form associated with mental retardation, microcephaly, nephrolithiasis, seizure disorder, accelerated atherosclerosis, marked increase in thromboembolic disease Heterozygous form assoc. with mildly increased thromboembolic disease but not other problems

HYPERCOAGULABLE STATES Hyperhomocysteinemia Homocysteine + Serine CystathioneCysteine CBS HomocysteineMethionine MTHFR

HYPERCOAGULABLE STATES Hyperhomocysteinemia - Causes Vitamin B 12 deficiency Folic acid deficiency Vitamin B 6 deficiency Cysthathione synthase deficiency (classic form) Methyl tetrahydrofolate reductase deficiency (most common by far)

HYPERCOAGULABLE STATES Hyperhomocysteinemia - Diagnosis Fasting homocysteine levels; considerable variability depending on assay Methionine loading if clinical suspicion high, but can precipitate thrombosis Methyl tetrahydrofolate reductase mutation (MTHFR C677  T) - Only relevant if homozygous

HYPERCOAGULABLE STATES Acquired Anticardiolipin Syndrome Malignancy Immobilization TTP DIC Oral Contraceptive Therapy Prosthetic Valves PNH Myeloproliferative diseases Nephrotic Syndrome Inflammatory Diseases Atherosclerosis Surgery Diabetes mellitus

ANTICARDIOLIPIN ANTIBODY Lupus Anticoagulant Not necessarily associated with lupus (< 50%) Not associated with bleeding except in rare circumstances Associated with thrombosis - arterial & venous Associated with false (+) RPR Associated with recurrent spontaneous abortions Mechanism of thrombotic tendency unknown

LUPUS ANTICOAGULANT Caused by antiphospholipid antibodies that interfere with clotting process in vitro but not in vivo Dilute phospholipid so level of phospholipid becomes rate-limiting Many add confirmatory study of either aPTT with platelets as PL source or orthogonal PL as PL source

ANTIPHOSPHOLIPID ANTIBODY Assay Usually antigenic as opposed to functional assay True antigen is source of controversy- ? if phospholipid is true antigen or if associated protein is true antigen ? Pathogenicity of what is being measured Impossible to standardize assay even batch- to-batch of reagents

ACQUIRED HYPERCOAGULABLE STATES Mechanisms in Acute Inflammation C4b Binding Protein - Acute Phase Reactant Increases in inflammatory diseases Binds to Protein S Bound Protein S inactive as cofactor Inflammation  Increased IL-1 & TNF Both downregulate thrombomodulin Thrombin becomes procoagulant instead of anticoagulant protein

PROTHROMBOTIC DISORDERS Summary No screening test readily available Probably look at genetic tests 1st Factor V Leiden Prothrombin G20210A MTHFR mutation Antiphospholipid antibody studies Homocysteine levels Protein C, Protein S, ATIII, Plasminogen Look for signs of inflammation Consider prolonged anticoagulant Rx if any of above positive Screen family for disease if positive